Before looking at the various strategies to study fatty acids you would
like to learn details about the history of their discovery, so read the
next chapter.

HistoryWe owe a considerable debt to ancient investigators who, prior to about
1935, made enormous contributions to our knowledge of the fatty acid composition
of natural lipids despite primitive equipments and analytical techniques. Since
the first works of Chevreul and for about a century, chemists isolated lipids
using only solubility properties of solvents, the formation of salts of fatty
acids which were further characterized by their raw formula, and ebullition or
fusion temperatures.
The period following 1935 has been marked by new and more efficient procedures
for separating and studying fatty acid mixtures. These procedures include ester
distillation, crystallization of urea complexes or of various metallic salts,
various forms of chromatography and countercurrent distribution.

The discovery in the mid-1950's of gas-liquid
chromatography (GLC) has revolutionized the analysis of fatty acids and, undoubtedly, this
technique is the most frequently used. Indeed, for the quantification of individual fatty
acids in any acylated lipids, GLC must be adopted.
In some other studies, complementary techniques should be considered. Metabolic studies
involve the knowledge of the intensity of labelling of molecular species with radioactive
atoms while identification studies require the separation and quantification of
hydroxylated, branched-chain, trans or conjugated fatty acids. All these investigations
are more easily run with HPLC than with GLC procedures since positional and conformational
isomers are more easily separated by HPLC than by GLC. Furthermore, HPLC is the method of
choice for preparative scale separations of particular fatty acids for further structural
or metabolic studies. In contrast to GLC which preferred flame ionization detection (FID),
the choice of the detector for HPLC analysis is important and determines the adopted
procedure. Several detections are possible, the most used are light scattering, UV,
fluorescence and radioactivity.
In general, fatty acids are separated by HPLC as derivatized molecules but unesterified
forms can also be chromatographed if acidic solvent systems are used.

For some precise purposes only the amount of fatty acids is to be known. Global
methods are useful when the fatty acid profile is not in the scope of the
investigation.

Fatty acids may be found in scarce
amounts in free form but, in general they are combined in more complex molecules
through ester or amide bonds.
The isolation of free fatty acids from biological materials is a complex task
and precautions should be taken at all times to prevent or minimize the effects
of hydrolyzing enzymes.

When fatty acids (medium and long-chain) are in aqueous media they may be accurately
extracted using a small C18 bonded phase column (SPE) (Battistutta F et al.,
J High Resol Chromatogr 1994, 17, 662). This method was also used to isolate
fatty acid ethyl esters from alcoholic beverages. Shortly, the SPE cartridges
are prepared in washing with methanol and water. 50 ml of liquid are passed
through the column followed by a washing with acidified water. Analytes are
eluted with 2 ml dichloromethane and 2.5 ml pentane.

The extraction of long-chain fatty acids from fermentation medium and industrial
effluents with a 98 to 100% recovery was described (Lalman JA et al., JAOCS
2004, 81, 105). Maximal recovery was obtained by adding 2 ml of hexane/ter-butyl
methyl ether (1/1), 80 ml
of 50% H2SO4,
and 0.05 g NaCl to 1 ml of the aqueous sample and mixing for 15 min at 200 rpm.
A lower recovery was obtained only for caproic (C6:0) and caprylic (C8:0) acids
: 27 and 76% recoveries, respectively.

The purification of free fatty acids has been done by solid-phase
microextraction (SPME) (Tomaino
RM et al. J Agric Food Chem 2001, 49, 3993). The fiber sheath of a 30
mm
thick poly(dimethylsiloxane) fiber (Supelco) was incubated at 110°C for 80 min
in the acidified medium and then placed into the injector of a gas chromatograph
whose temperature was increased from 100°C to 245°C. Unfortunately, a progressive
and rapid loss of sensitivity occurred with decreasing fatty acid chain length.
Thus, it was necessary to determine the response factors for each fatty acid
in relation to an internal standard (C17). Advantages of that extraction procedure
are the little sample preparation, the absence of organic solvents, the detection
of short chain fatty acids, and a good reproducibility.

A one-step extraction and derivatization method has been proposed, essentially
based on a dispersive liquid-liquid microextraction (Pusvaskiene E et al.,
Chromatographia 2009, 69, 271). This simple and fast method using ethyl
chloroformate as derivatization reagent was applied for the determination of
free fatty acids in water (tap, lake, sea, river).
For many years, diazomethane was the reagent of choice to selectively derivatize
and then detect free fatty acids due to its highly specific methylation of the
carboxylic acid functional group. While its activity is very defined, it is
dangerous and can be difficult to obtain. An important review has compiled a
collection of methods which allow for the detection of hydroxy and non-hydroxy
free fatty aicds without the use of diazomethane (Potter G et al., Eur J
Lipid Sci Technol 2015, 117, 908).

A convenient, economic, and high throughput approach has been established to
separating free from esterified fatty acids in using a chemical derivatization
and immobilization on amino silica nano-paarticles (Chen
J et al., J Chromatogr A 2016, 1431, 197).

Short-chain fatty acids (C1 to C5) in biological specimens need a special treatment
taking into account their volatility. Thus a simple and efficient procedure
using a vacuum transfer followed by HPLC enable the accurate determination of
these acids in the nanomolar range in tissues and secretions (Stein J et
al., J Chromatogr 1992, 576, 53). An eficient procedure using an extraction
with a hollow fiber coupled with gas chromatography has been reported (Zhao
G et al., J Chromatogr B 2007, 846, 202).
Application of gas chromatography coupled to mass spectrometry following headspace
solid-phase microextraction was applied with great accuracy and sensitivity
to the determination of free volatile fatty acids in aqueous samples (Abalos
M et al., J Chromatogr A 2000, 891, 287). Valuable results were obtained
for the determination of C2-C7 fatty acids in raw sewage.
Free medium-chain fatty acids in beer have been extracted using adsorption on
a specific stir bar (Gerstel
twister). The determination of caproic, caprylic, capric and lauric acids
with solvent back extraction was described (Horak
T et al., J Chromatogr A 2008, 1196-1197, 96). The procedure utilized
10ml of sample stirring with the stir bar with 1000rpm for 60min at room temperature.
Solvent back extraction used 200ml
of solvent (dichloromethane/hexane, 50/50) at room temperature.

Bound
fatty acids

When fatty acids are combined in
more complex molecules such as acylglycerols, cholesterol esters, waxes and
glycosphingolipids, they can be obtained free by saponification (inorganic
or organic basic solution) or acidic hydrolysis and then derivatized.
FAME may be also obtained directly by transesterification (alcoholysis or methanolysis)
of the fatty acid-containing lipids.The
extraction and methylation may also be combined in a one-step procedure,
this is particularly recommended for very small samples in order to prevent
any loss of fatty acids during the classical procedures.A
usefull comparison of the various derivatization methods my be consulted (Ostermann
A.I., et al., Prostagl, Leukotr Essential Fatty Acids 2014, 91, 235). A
detailed protocol for the analysis of plasma and tissues is included in this
article.

Saponification

When fatty acids are required in
free form for further analysis, lipids (present as glycerides, glycerophosphatides,
glycosyldiglycerides, sterol esters or waxes) are first hydrolyzed in alkaline
medium allowing to extract also the unsaponifiable material if present in the
crude lipid mixture (sterols, alcohols, hydrocarbons, pigments, vitamins...).
Glycosphingolipids are poorly hydrolyzed with the described procedure but, if
any contribution of these complex lipids is to be avoided, a
mild saponification process must be adopted.

Pipet an aliquot of lipid extract
(up to 30 mg) into a screw-capped tube (Teflon-lined). Evaporate the solvent
and add 5 ml methanolic KOH. Warm for 1 h at 80°C in a water or a sand bath.
After cooling, extract the non-saponifiables with 2 washings of 5 ml diethyl
ether. Add a few drops of phenolphthalein indicator to the lower phase and acidify
with HCl (about 0.3 ml).
Extract the fatty acids with 2 washings of 5 ml hexane. When short-chain fatty
acids are present in the lipid extract, it is necessary to extract more extensively
with hexane (5 or 6 times). Do not evaporate too extensively the hexane phase
(keep at a mild temperature) to prevent
loss of these fatty acids.
Fatty acids may be weighed, titrated to determine their neutralization equivalent
or converted to methyl esters before fractionation
or GLC analysis..

An alternative method for saponification
has been proposed using a microwave-assisted treatment (Pineiro-Avila G et
al., Anal Chim Acta 1998, 371, 297). A closed reactor containing the lipid
sample and an adapted volume of ethanolic KOH solution is irradiated for a short
time (2-3 min) in a microwave oven at an exit power of about 350 W. The extraction
of fatty acids is then processed as described above.

Saponification of dry powder may be done directly before the extraction
of fatty acids or non-saponifiable compounds (Sanchez-Machado
DI et al., J Chromatogr A 2002, 976, 277).
250 mg of ground samle are mixed with 5 ml of 0.5M KOH in methanol. The tubes
are incubated at 80°C for 15 min (vortexing every 5 min). After cooling in ice,
1 ml water and 5 ml hexane are added and the tubes are vortexed for 1 min. After
a short centrifugation, 3 ml of the upper phase are transferred to another tube
and dried under nitrogen before analysis.

Acidic hydrolysis

When the investigated lipid extract
contains complex lipids as sphingolipids, an efficient procedure to free amide-bond
fatty acids is needed. It is recommended to fractionate any crude lipid extract
into glycerolipids and glycosphingolipids before applying an alkaline saponification
to the former and an acidic hydrolysis to the later.
The procedure previously proposed for ceramides
consists in a treatment with methanolic HCl in presence of water which is known
to give rise to only minor amounts of by-products. It is noticeable that this
procedure yields directly FAME ready to be fractionated or analyzed by GLC.

Organic basic hydrolysis

The organic basic solution, 1 M tetramethylammonium hydroxide (TMAH)
was employed and recommended for the hydrolysis of extremely small amounts of
lipids (lower than 1 mg) (Woo
KL et al., J Chromatogr A 1999, 862, 199). That procedure was found
excellent for small samples while saponification with ethanolic KOH was found
unsuitable. Using TMAH, a 2 fold recovery of long-chain fatty acids was obtained
as compared with the classical KOH hydrolysis and the reliability of data was
very high.

Deacylation of cerebrosides and sulfatides by a powerful microwave-mediated
saponification was reported (Taketomi T et al. Biochem Biophys Res Comm 1996,
224, 462). The reaction was run in 0.1 M NaOH in methanol for 2 min in 500W
microwave oven. After acidification the fatty acids are extracted in hexane
and methylated.

Combined basic and acid hydrolysis

Another practical approach to the technical
problem of the hydrolysis of sphingolipids has been described using a one-spot
heating in a microwave oven with 0.1 M NaOH in methanol for 2 min followed by
1M HCl in methanol for 45 s (Itonori
S et al., J Lipid Res 2004, 45, 574).

DERIVATIZATION
BEFORE GLC

Before GLC
analysis it is necessary to prepare non-reactive derivatives of fatty acids
(methyl esters or other derivatives) which are also more volatile than the free
acid components. Acylated lipids are transformed by a transesterification reaction
by which the glycerol moiety is displaced by another alcohol (methanol, butanol,
propanol...) in acidic conditions (HCl or BF3).

The generation of methyl esters can be done in acidic or in alkaline
conditions on isolated lipids or fatty acids but also directly by a one-step
procedure combining lipid extraction and transesterification on small amounts of dried
tissue.
On a large scale, fatty acid methyl esters, used as a substitute of diesel fuel
(Biodiesel), are prepared by
transesterification of vegetal oils with sodium methylate, NaOH or KOH in dry
medium.

Other fatty acid derivatives may be prepared as an
answer to some specific problems

A - Acid-catalyzed esterification

The most common derivatives of fatty acids are
the methyl esters obtained by heating free fatty acids with a large excess of anhydrous
methanol in the presence of a catalyst, boron trifluoride (Morrison et al J Lipid Res
1964, 5,600). It must be noticed that O-acyl lipids are transesterified very rapidly
with the same reagent.

Reagent

14% Boron trifluoride in methanol (Alltech or
Sigma) (keep refrigerated under nitrogen and discard after 3 months or when solids appear
at the bottom of the vial).
Pentane, chloroform.

Procedure

As a general procedure, an aliquot of lipid
extract (about 10 mg) is dried under nitrogen in a screw-capped glass tube and 1 ml of BF3/methanol
is added.
If triacylglycerols or sterol esters are analyzed alone or are abundant in the extract,
the dry lipids are dissolved in 0.75 ml of chloroform/methanol (1/1, v/v) and 0.25 ml BF3/methanol
are added. If possible, the tube is closed after flushing with nitrogen.
Heat in boiling water (or at 100°C in a sand bath) the time indicated for the respective
lipid:

Lipids

Heating time (min)

Fatty acids

5

Triacylglycerols

45

Sterol esters

45

Monoacylglycerols

15

Diacylglycerols

15

Glycerophospholipids

15

Glyceroglycolipids

15

Sphingomyelin

90

Glycosphingolipids

90

After cooling, add 1 ml water
and 2 ml pentane. Vortex for 1 min, centrifuge at low speed and collect the upper phase.
Pentane is evaporated and the residue is immediately dissolved in 50-100 µl hexane. The
solution is ready for injection in the gas chromatograph.After TLC, spots containing fatty acid-based
lipids may be scraped, collected and treated with the BF3/methanol solution
directly in a glass tube. It was reported that selective loss of unsaturated
fatty acids was observed oon certain brands of plates (Sowa JM et al., J
Chromatogr B 2004, 813, 159). Thus, the authors determined that no loss
occurred in both neutral and phospholipids with Alltech or Merck silica gel
plates.

Preparation of fatty acid methyl esters from various sources using commercial
aqueous HCl was also described (Ichihara
K et al., J Lipid Res 2010, 51, 635). Yields of FAME were the same as
those obtained with boron trifluoride method. Furthermore, the reagent is very
convenient, safe and cheap.
Shortly, the reagent is made from 9.7 ml commercial concentrated HCI (35%, w/w)
diluted with 41.5 ml of methanol and was stored in a refrigerator. A lipid
sample was dissolved in 0.20 ml of toluene, then 1.50 ml of methanol and 0.30 ml
of the reagent solution were added in this order. The tube was vortexed and then
heated at 100°C for 1 h. After cooling, 1 ml of hexane and 1 ml of water were
added for extraction of methyl esters in the hexane phase.

An improved method for determining medium- and long-chain in lipid samples using
one-step transesterification with acetyl chloride has been reported (Xu Z et
al., Lipids 2010, 45, 199). The data suggest that the method can be easily
used to accurately determine fatty acids (C6–C24) in functional foods and
lipid emulsions.

B - Base-catalyzed
transesterification

Fatty esters form with a base (alcoholate) form an
anionic intermediate which is transformed in the presence of a large excess of
the alcohol into a new ester. Free fatty acids are not subject to nucleophilic
attack by alcohols or bases and thus are not esterified in these conditions.
Derivatizations in the presence of basic catalysts
have the advantages of speed and mild heating conditions. Thus this type of
catalysis is recommended in samples with short-chain fatty acids or labile fatty
acids (polyunsaturated, cyclopropane rings, conjugated unsaturations...).

The most useful basic transesterifying agents are 1 to 2M Na or K methoxide in
anhydrous methanol. These solutions are stable for several months at 4°C until
a white precipitate of bicarbonate salt is formed. Glycerolipids are rapidly
transesterified (2-5 min) at room temperature.

Up to 10 mg of lipids are dissolved in 2 ml hexane followed by the addition
of 0.2 ml of 2 M methanolic KOH. The tube is vortexed for 2 min at room
temperature. After a light centrifugation, an aliquot of the hexane layer is
collected for GC analysis.
It must be pointed out that sterol esters and waxes do not react under these
conditions.

A modification and an adaptation of that procedure has been proposed allowing
the direct preparation of fatty acid methyl esters from polar lipids
(phospholipids) in lipid mixtures without prior isolation (Ichihara K et
al.,
Lipids 2010, 45, 367). No obvious differences were found between the fatty
acid compositions of phospholipids determined by that method and those
determined by conventional methods, including lipid extraction with
chloroform/methanol followed by isolation of polar lipids by chromatography.

100 ml
of a solution of K tert-butoxide in THF are added to 200 ml
anhydrous 2-methoxyethanol in a closed vial. After homogenization, up to 10 mg
of lipid in 1 ml hexane are added. Keep the mixture at 40°C for 15 min. After
cooling, 1 ml water and 2 ml hexane are successively added. After 5 s vortexing
and a short centrifugation, the organic phase is collected, dried over anhydrous
Na sulfate and analyzed by GLC.

We have adopted another
approach for some labile samples. A rapid and mild method which avoids the formation
of oxidation products was described by Piretti et al. (Chem Phys Lipids 1988,
47, 149). We have most precisely adopted this procedure for the analysis
of highly unsaturated lipids since higher amounts of polyunsaturated fatty acids
were found when compared to the BF3/methanol procedure.
Furthermore, if hydroperoxy fatty acids are present, they are reduced into the
corresponding hydroxy components.

Reagents:

2M NaOH, NaBH4, anhydrous
Na2SO4.
Ethyl acetate, methanol, hexane.

Procedure:

2 mg of neutral lipids or up to 100
mg polar lipids are dried in a glass tube.
Add 1 ml of the reagent made in dissolving immediately before use 400 mg NaBH4
in 10 ml of the mixture methanol/2 M NaOH (19/1, v/v).
The mixture is stirred for 20 min at room temperature. After adding 2 ml water,
the methanol is eliminated under nitrogen. The methyl esters are recovered from
the aqueous phase by extracting 3 times with 1 ml ethyl acetate. The organic
phase is then washed 3 times with 1 ml water and dried by adding Na2SO4.
After vortexing and centrifugation, the ethyl acetate is evaporated and the
residue dissolved in a small amount of hexane for GLC analysis.

A one-step method with saponification
followed by 60 min of methylation time has been described as a simple, fast
and accurate tool to quantitatively analyse fatty acids in human red blood cells
(RBC) for for clinical and nutritional studies (Rodrigues RO et al., Chromatographia
2015, 78, 1271).Shortly: In a 7 mL glass
vial, 150 µL of RBC sample containing internal standard and BHT were added
and mixed with 500 µL of methanolic KOH solution (0.2 M). Vials were capped
and vigorously vortexed for 30 s followed by a nitrogen flushing. Saponification
was performed at 90 °C during 10 min. After cooling the samples, 2 mL of
BF3 methanolic solution was added and the vials were vortexed for 30 s followed
by nitrogen flushing. Transmethylation was performed at 90 °C during 60
min. After cooling the samples, 1 mL of n-heptane was added and fatty acid methyl
esters were extracted twice by vortex mixing
(30 s). The supernatant was transferred to a clean glass vial and evaporated
under nitrogen gas. Finally, samples were resuspended in 100 µL of n-heptane
and analysed by GLC.

Another one step and very
rapid 10 seconds) method has been described using sodium methylate as the main
reagent for the analysis of triacylglycerol composition (Ichihara
K et al., Anal Biochem 2016, 495, 6).Shortly,
methanolic CH3ONa (2 M) is prepared by diluting 25% (w/w, 4.37 M) CH3ONa with
methanol. In a small glass test tube are placed 1 ml of hexane containing 20
mg or less of triacylglycerols and 0.5 ml of acetone. To the lipid solution
is added 75 ml of 2M CH3ONa with vortexing. Methanolysis is completed within
10 s and the reaction is terminated with 1 ml of 0.5 M acetic acid.The
upper organic layer containing methyl esters is washed with 1 ml of water. The
hexane solution of FAMEs is then analyzed by gas chromatography.Under
these conditions, trioleoylglycerol is converted to methyl oleate with an average
yield of 99.3%.

C - Direct transmethylation
without prior extraction

The concept of direct transesterification of techniques
has been reported for small tissue samples (1-10 mg) or small volumes (about
50 ml)
of biological fluids (blood, milk) and plant samples.

Procedure for small tissue samples :

A tissue sample containing as low as 10 mg
of lipids is introduced at the bottom of a screw-capped tube (Teflon-lined). Then add 1 ml of methanolic HCl, 1 ml of methanol and 0.5 ml hexane. Close
tightly the tube and heat at 100°C for 1 h (shake several times).
After cooling add 2 ml of hexane and 2 ml of water. Mix not too vigorously the
tube and collect the hexane layer after a short centrifugation. Before GC
analysis, the extract may be concentrated by evaporation under nitrogen if
necessary.

Comments :

The total fatty acid composition of plasma was determined with a
transesterification procedure similar to that described above (Glaser C et al.,
PlosOne 2010, 5, e12045). As claimed by the authors, the sample preparation time
and analysis costs are reduced to a minimum. The method is an economically and
ecologically superior alternative to conventional methods for assessing plasma
fatty acid status in large studies.

In lipid-producing bacteria or microheterotrophs, the direct transesterification method was shown to
be the most efficient to study the fatty acid profiles (Lewis T et al. J
Microbiol Meth 2000, 43, 107). The proposed procedure consists in
treating freeze-dried cells at 90°C for 60 min in the mixture methanol/conc
HCl/chloroform (10/1/1, v/v)(3 ml). After addition of water (1 ml), fatty acid
methyl esters are extracted by vortexing 3 times with 2 ml of hexane/chloroform
(4/1, v/v).

A critical review on in situ transesterification avoiding the use of
lipid extraction describes all aspects in order to achieve accurate and reliable
results (Carrapiso
AI et al. Lipids 2000, 35, 1167). An application of direct
transmethylation to red blood cell membranes and cultured cell has been also
described (Rise P et al., Anal Biochem 2005, 346, 182).
A comparative study of "direct" and "two steps" (extraction
followed by derivatisation) methods has been done with plasma samples (Amusquivar
E et al., Eur J Lipid Sci Technol 2011, 113, 711). The ‘‘two steps’’
method appears more appropriate and reliable, and C19:0 but not C15:0 should be
used as the internal standard.

A quantitative and simple in situ method for the assessment of the fatty acid
composition of solid samples (triturated seeds, lard, muscle) through their
pentyl esters was described (Eras J et al., J Chromatogr A 2004, 1047, 157).
The reaction was carried out using chlorotrimethylsilane and 1-pentenol as
reagents for 40 min at 90°C. It permits major recoveries of the total
saponifiable lipids present in solid samples, a 40 min reaction time ensuring
the total conversion of lipids to the corresponding fatty acid pentyl
esters.
A similar but more rapid (30 s) transesterification process using a one step
carried out in a microwave reactor has been described for quantifying meat
acylglycerides (Tomas
A et al., J Chromatogr A 2009, 1216, 3290).

A comparative study between the direct methylation and the
classic procedure has shown that, in eggs, the direct methylation procedure was
less precise than the second procedure (Mazalli
MR et al., Lipids 2007, 42, 483).

A rapid and efficient method for direct transesterification of lipids from plant
sources has been described and compared with several other derivatization
procedures (Alves SP et al., J Chromatogr A 2008, 1209, 212). The most
efficient procedure was as follow : 1mL of
internal standard (C17:0, 1mg/mL) and 1mL of toluene were added to 250mg of
sample, followed by the addition of 3mL of 5% HCl solution in methanol (prepared
by the addition of acetyl chloride to the methanol). After homogenization on
vortex at slowspeed, sampleswere maintained for 2h at 70◦C in a water
bath. After that, the solution was left to cool at room temperature and
subsequently neutralized with 5mL of 6% K2CO3. FAMEs were extracted with 2mL of
hexane, and 1 g of both Na2SO4 and activated carbon were added. Finally, samples
were centrifuged for 5 min at 2500 rpm, the supernatantwas transferred to new
tubes and the solvent removed under nitrogen at 37 ◦C. The final residue
was dissolved in 1mL of hexane, and stored until GC analysis.
An additional step based on solid-phase extraction was necessary to produce
clean samples.

An efficient direct transesterification has
been described for assay of the fatty acid content of microalgae (Griffiths
MJ et al., Lipids 2010, 45, 1053). Higher levels of fatty acid in the
cells were obtained with that procedure in comparison with the extraction-transesterification
methods. A combination of acidic and basic transesterification catalysts was
more effective than each individually when the sample contained water. The
two-catalyst reaction was insensitive to water up to 10% of total reaction
volume.

A micromethod for the fatty acid analysis of glycerophospholipids using a sodium
methoxide solution has been described with cheek cell samplings (Klingler
M et al., Lipids 2011, 46, 981-90).

Procedure for
small amounts of bacteria :

The knowledge of the fatty acid composition of microorganisms is now
recognized as essential for their taxonomic classification as well as for the
evaluation of the nutritional quality of alternative microbial sources of fats.
To guarantee a high recovery of fragile fatty acids, such as cyclopropane and
conjugated linoleic acids, as well as a high degree of methylation for all types
of fatty acids, a rapid and reliable method is needed. A direct methylation
method representing a valuable alternative to other methylation procedures has
been described (Dionisi F et al., Lipids 1999, 34, 1107).

Procedure :

One hundred milligrams of dried bacterial samples, with 500 mg
of internal standard, is transesterified using 1 ml of methanolic HCl (1.5M)
(from Supelco) and 1 ml methanol, at 80°C for 10 min. Water (2 ml) is added and
after mixing and low speed centrifugation the upper phase is collected for gas
chromatographic analysis.

Procedure for small amounts of
fluid :

A convenient method was developed for preparation of fatty acid methyl esters
in glycerolipids of blood or milk (Ichihara K et al., Lipids 2002, 37, 523).

Procedure:

About 50 ml
of blood or milk are spotted onto a small piece of Whatman 3MM filter paper
(1.5x1.5 cm) that has been previously washed with acetone containing 0.05 %
BHT. Each piece, once dried for 30 min in vacuo is inserted into a small test
tube, to which 2 ml hexane and 0.2 ml 2M KOH/methanol are added (alkali-catalyzed
alcoholysis). After vigorous mixing or sonication for 2 min at room temperature,
the solution is neutralized with acetic acid. To each tube is added 2 ml water
with light mixing. An aliquot of the hexane layer was collected and evaporated
to dryness; FAME are dissolved in 0.02 ml hexane or methyl acetate before GC
analysis.
The presence of BHT on the filter paper allows the protection of unsaturated
fatty acids for at least 7 days even exposed to the air.

A similar direct procedure using boron trifluoride-methanol as esterification
reagent was described for the determination of fatty acids in human milk (Lopez-Lopez
A et al., Chromatographia 2001, 54, 743).

A direct evaluation of the fatty acid status in a drop of blood was described
(Marangoni
F et al., Anal Biochem 2004, 326, 267). No more than 50 ml
of blood were absorbed on a piece of chromatography paper and directly treated
with 3 N methanol/HCl at 90°C for 1 h. The method was validated for reproducibility
and satisfactorily compared with a conventional method.

Procedure for dried samples

A convenient method was developed for
preparation of fatty acid methyl esters directly on freeze-dried milk
samples. The extraction step is not required and the sample can be immediately
subjected to the transesterification procedure (Gastaldi D et al., Chromatographia
2009, 70, 1485).
Briefly, a volume of 60 ml
of a 500 mg
per ml standard solution of linoleneaidic acid (internal standard) was evaporated
to dryness under nitrogen in a 20 ml centrifuge tube provided with a Teflon-lined
screw cap. Weighed amounts of sample were added to the residue. 3 ml of boron
trifluoride–methanol reagent were added to the mixture under nitrogen. The tube
was closed, heated at 80 °C for 45 min and cooled. For fatty acids methyl esters
extraction, 1 ml of a NaCl saturated aqueous solution and 3 ml of n-hexane were
added. The mixture was vigorously shaken and phase separation was achieved by
centrifugation. 1.5 ml of clear supernatant was transferred into an autosampler
vial for GLC analysis.

A very precise study was developed to analyze fatty acids in a capillary dried
blood spot system in order to protect n-3 long-chain fatty
acids from oxidation for up to 2 months at room temperature. The methodology
has been validated for clinical applications through a direct comparison with
established methods (Liu G. et
al., Leukotr Essential Fatty Acids 2014, 91, 251).

D - Other fatty acid derivatives

Butylation - Propylation

Methylation is not efficient for analyzing
carboxylic acids of medium or short chain (< C12) as their volability can
lead to unquantifiable losses. Thus, derivatizations forming propyl or butyl
esters have been used for a long time. Butyl esters are more frequently used
for simultaneous analysis of low- and high-molecular weight fatty acids. The
conversion efficiency of various carboxylic acids has been reported under different
reaction conditions (Hallmann
C et al., J Chromatogr A 2008, 1198-1199, 14). The most efficient recovery
for fatty acids was obtained using n-butanol/BF3 (10%, w/w) from
from Sigma–Aldrich at 100°C for 2 hours. Care must be taken when different types
of carboxylic acids are to be analyzed.

The recovery of short-chain fatty acids in milk fat is improved when the analysis
of the fatty acid composition by gas chromatography is conducted with the propyl
derivatives, instead of the methyl esters (Sasaki R et al., J Oleo Sci 2015,
64, 1251). In this study, with the aim to identify minor fatty acids, the
propyl esters were fractionated by Ag-ion solid phase extraction before gas
chromatography. Silylation

If methyl esterification with BF3/methanol
has been the most widely used derivatization method, other approaches were described
to correct various defects such as reagent instability, destruction of epoxy,
cyclic fatty acids, hydroxy groups, and non-derivatization of unsaponifiable
materials. Trimethylsilyl derivatization is known to be an efficient method
but it has some faults like thermal instability and partial hydrolysis of the
derivatives. To overcome these defects, the ter-butyldimethylsilyl (tBDMSi)
derivatization method for GC analysis was developed (Woo KL et al., J Chromatogr
A 1999, 862, 199). These derivatives were shown to have a high thermal and
hydrolytic stability and they improve the sensitivity and the selectivity of
the analyses.

Procedure :

To fatty acids dissolved in 200 ml
of hexane, a known amount of internal standard solution, 75 ml
of N-methyl-N-(ter-butyldimethylsilyl)trifluoroacetamide and 5 ml
of triethylamine are added. After tightly capping, the contents are maintained
at 75°C for 30 min before injection.
The separation is done with a HP-1 capillary column (50 m x 0.2 mm ID) with a
temperature program as follows : 40°C for 1 min and then after increasing to
70°C with 60°C/min, held for 2 mn. After increasing to 205°C with 5°C/min,
held for 25 min and then increased to 285°C with 5°C/min and held for 1 min.
Injector and detector are at 300°C.

Comments :

In all fatty acids, the peak responses for these derivatives are higher by
1.5-6.3-times than for methyl esters. In contrast, the stability was shown to be
reduced practically to no more than 3 days.

Cyanomethylation

During cyanomethylation the
carboxyl group of fatty acids is alkylated to cyanomethyl esters (R-COO-CH2-CN)
and derivatives are detected with nitrogen-phosphorus detector. The method is
rapid, inexpensive, and resistant to contaminants frequently found during the
chromatographic separation of very-long-chain fatty acids (Paik MJ et al., J
Chromatogr B 1999, 721, 3).